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Synthesis of Locust Bean Gum new
derivatives and their application in
nanoparticulate drug delivery systems
Luis Braz
D
2016D
.FFUP
2016
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Luis Manuel Lima Verde de Braz
Synthesis of Locust Bean Gum new derivatives
and their application in nanoparticulate drug
delivery systems
Thesis submitted in fulfilment of the requirements to obtain the PhD degree in Pharmaceutical Sciences,
Pharmaceutical Technology Speciality, Faculty of Pharmacy of University of Porto
Work developed under supervision of Prof. Dr. Bruno Sarmento and co-supervision of Prof. Dr. Ana M Rosa da Costa and Prof. Dr. Domingos de Carvalho Ferreira
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The full reproduction of this thesis is allowed for research purposes only, through a written declaration of the person concerned, to which he commits to.
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“Don’t count the days, make the days count.”
Muhammad Ali
To Guida, Diogo and Inês
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helped and supported me during this work. Thus, I would like to acknowledge:
My supervisor Professor Bruno Sarmento and my co-supervisors Professor Domingos Ferreira and Professor Ana Costa for all the support during the development of this work and thesis.
Professor Bruno Sarmento, my supervisor, from Instituto de Engenharia Biomédica (INEB) and Instituto de Investigação e Inovação em Saúde (I3S) da Universidade do Porto, Porto, Portugal, and from Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde (IINFACTS) do Instituto Superior de Ciências de Saúde do Norte (ISCS-N) da Cooperativa de Ensino Superior Politécnico e Universitário (CESPU), Gandra Portugal, for accepting me as a PhD student, for the support and scientific guidance during the development of this work and the writing of the thesis. I also acknowledge his patience and, most of all, friendship.
Professor Domingos Ferreira, my co-supervisor, from Laboratório de Tecnologia Farmacêutica da Faculdade de Farmácia, Universidade do Porto (FFUP), Porto, Portugal, for the support, friendship, understanding and availability provided to make possible the development of this work.
Professor Ana Costa, my co-supervisor, from Centro de Investigação em Química do Algarve (CIQA), Universidade do Algarve, Faro, Portugal and from Faculdade de Ciências e Tecnologia, Universidade do Algarve (FCT-UAlg), Faro, Portugal, for accepting the challenge of being my PhD co-supervisor, for all the support and scientific guidance during the development of this work and the writing of the thesis. Also for her patience, for being very present during the bench work, especially during the challenging chemical modifications performed in locust bean gum. Thank you also for sharing with me the most difficult and most exultant moments during the performance of this work, and for her dedicated friendship.
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Professor Ana Grenha from Centro de Investigação em Biomedicina (CBMR) da Universidade do Algarve, Faro, Portugal and from Centro de Ciências do Mar (CCMAR) da Universidade do Algarve, Faro, Portugal and from FCT-UAlg, for all the support given during the development of this work, especially for lending a space in her laboratory and allow me to use her material and reagents.
All the colleagues at CBMR, particularly Susana Rodrigues for all the friendship and support during the development of this work. Thank you for feeding the cells and seeding the plates when I was unable to be there.
Professor João Lourenço from CIQA, FCT-UAlg and Centro de Química Estrutural (CQE), Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal for assistance in the X-ray diffraction analysis.
Professor Marta Covo from Research Unit on Applied Molecular Biosciences (UCIBIO) at Rede de Química e Tecnologia (REQUIMTE), Departmento de Química, and CENIMAT/I3N, Departmento de Ciência de Materiais, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal, for assistance in the NMR analysis.
Professor Carlos Gamazo from Departamento de Microbiología y Parasitología da Universidad de Navarra (UNAV), Pamplona, Spain, for receiving me in his group at UNAV during my short stay, and for all the support in the in vivo studies performed there. Thank you for the short moment during all this time when I really felt like a PhD student.
Ana Camacho (now Professor Ana Camacho, congratulations!) from Departamento de Microbiología y Parasitología da Universidad de Navarra (UNAV), Pamplona, Spain, for assistance during the in vivo experiments performed in UNAV.
Marinella from CBMR for assistance in the day 0 blood sample collection in the in vivo assays performed in UAlg.
Patrícia Madureira from CBMR for assistance in the SDS-PAGE and immunoblotting analyses performed in UAlg.
All my colleagues at Escola Superior de Saúde da Universidade do Algarve (ESSUAlg) for their support and friendship.
ix just for the records!).
This work was supported by National Portuguese funding through FCT – Fundação para a
Ciência e a Tecnologia, projects PTDC/SAU-FCF/100291/2008,
PEst-OE/EQB/LA0023/2013, PEst-OE/QUI/UI4023/2011 and UID/Multi/04378/2013. PROTEC grant from Direção Geral do Ensino Superior (SFRH/PROTEC/67422/2010), and mobility grant from ShareBiotech are also acknowledged.
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biopharmaceuticals, including for vaccination purposes. In this respect, they should focus on optimizing antigen association efficiency, provide stability, tailor the release and elicit high levels of long-lasting antibody and cellular immune responses. Nanoparticles may benefit oral immunization due to the predominant uptake of particulates by Peyer patches. The M cells have been pointed as the primary targets to consider for nanoparticles. After nanoparticle uptake, subsequent internalization by professional antigen presentation cells is expected to occur, mediating the following immune response. Additionally, nanoparticle matrix materials might further help on the potentiation of an immune response, and the use of mucoadhesive polymers, the surface chemistry and/or surface ligand conjugation play an important role. Locust bean gum (LBG) may contribute in a strong manner for the improvement of nanoparticle abilities regarding an application in oral immunization, as the chemical composition of this polysaccharide includes mannose residues that may provide a preferential targeting of M cells and/or dendritic cells.
The development of LBG-based nanoparticles for an application in oral immunization was, thus, proposed in this thesis. Nanoparticle production occurred by mild polyelectrolyte complexation, requiring the chemical modification of LBG. Three LBG derivatives were synthesized, namely a positively charged ammonium derivative (LBGA) and negatively charged sulfate (LBGS) and carboxylate (LBGC) derivatives. Glycidyltrimethylammonium chloride was the alkylating agent allowing to obtain LBGA, a N,N-dimethylformamide sulfur trioxide (SO3DMF) complex was the sulfating agent in the synthesis of LBGS, and
2,2,6,6-tetramethylpiperidine-1-oxyl the oxidizing agent used to produce LBGC. The derivatives were characterized by Fourier transform infrared spectroscopy, elemental analysis, nuclear magnetic resonance spectroscopy, gel permeation chromatography and x-ray diffraction. Since a pharmaceutical application was aimed, a toxicological analysis of the derivatives was required. The assessment of the metabolic activity of intestinal Caco-2 cells following exposure (3 h or 24 h) to LBG and derivatives was performed by the MTT test, demonstrating the general safety of LBG derivatives at concentrations up to 1.0 mg/mL, with the exception of LBGA. Similar observations resulted from a complementary cytotoxicity assessment evaluating cell membrane integrity (LDH release assay).
Several nanoparticle formulations were produced using LBGA or chitosan (either in the free amine, CS, or in the hydrochloride salt form, CSup) as positively charged polymers, and LBGC or LBGS as negatively charged counterparts. The nanoparticle formulations were obtained with production yields up to 58%, while sizes varied between 180 and 830
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nm and zeta potential between -28 mV and +48 mV, depending on the qualitative and quantitative composition. Morphological characterization performed on chosen formulations (LBGA/LBGS and CSup/LBGS) by transmission electronic microscopy suggested that nanoparticles presented a solid and compact structure with spherical-like shape. CSup/LBGS nanoparticles, which were later selected for the subsequent stage of antigen association, demonstrated to be stable in suspension for at least 3 months when stored at 4 ºC. LBGA/LBGS and CSup/LBGS nanoparticle formulations induced high cell viability in Caco-2 cells after 3 h and 24 h of exposure, when tested at concentrations up to 1.0 mg/mL (MTT assay), which was a remarkable event particularly considering the observation of some toxicity of the bare LBGA derivative. The LDH release assay evidenced some cytotoxicity of the CSup/LBGS formulation (24 h; 1.0 mg/mL), not shown by the MTT assay.
Two model antigens (a particulate cellular extract of Salmonella Enteritidis HE, and a soluble antigen - ovalbumin, OVA) were associated to CSup/LBGS nanoparticles with efficiency around 30%. The process was verified to not induce any deleterious effect on antigen structural integrity, while the antigenicity was retained. Nanoparticles exhibited adequate physicochemical properties for an application in oral immunization (size of 180 – 200 nm; positive zeta potential of 10 – 13 mV) and demonstrated to restrain the release of the antigens. Regarding the latter, a very limited release of HE in both simulated gastric and intestinal fluids was observed, while OVA released a maximum of 40% in the former medium. In vivo studies encompassed the administration of either HE-loaded or OVA-loaded nanoparticles to BALB/c mice. During five (HE) or six (OVA) weeks after oral and subcutaneous immunization, the systemic (IgG1 and IgG2a) and mucosal (IgA) immunological responses were evaluated. The adjuvant effect of the CSup/LBGS nanoparticles in obtaining an immunological response after oral immunization was demonstrated, although this was only provided when the soluble antigen OVA was used. On the contrary, an absence of effect was observed when the particulate antigen HE was tested. Nanoparticles were further found to elicit a balanced Th1/Th2 immune response, which is a relevant observation regarding an effective immunological protection.
Overall, LBGS was the synthesized derivative showing better ability for complexation with chitosan regarding the objective of producing nanoparticles with adequate properties for oral immunization. Additionally, a preliminary indication on the potential of the system for oral immunization is provided, although this is dependent on the antigen type.
Keywords: Locust bean gum, oral immunization, ovalbumin, polymeric nanoparticles,
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de biofármacos, incluindo em vacinação. Neste âmbito devem focar a otimização da eficiência de encapsulação, proporcionar estabilidade, modular a libertação e induzir níveis elevados e duradouros de resposta imunológica humoral e celular. As nanopartículas podem beneficiar esta abordagem devido à captura predominante de material particulado pelas placas de Peyer. As células M têm sido apontadas como principais alvos a considerar e, após internalização, é expectável a captura subsequente por células apresentadoras de antigénios profissionais, mediando a resposta imune que se segue. Adicionalmente, a matriz das nanopartículas pode potenciar a resposta imune e a utilização de polímeros mucoadesivos, com a sua química de superfície eventualmente aliada à conjugação superficial de ligandos, têm um papel importante. A goma de alfarroba (LBG) pode contribuir fortemente para melhorar a aplicação das nanopartículas em imunização oral, porque a sua composição química inclui resíduos de manose que podem proporcionar uma vetorização para as células M e/ou dendríticas. O desenvolvimento de nanopartículas de LBG para imunização oral é assim proposta nesta tese. A produção das nanopartículas ocorreu por complexação polieletrolítica, requerendo a modificação química da LBG. Três derivados foram sintetizados, um derivado aminado carregado positivamente (LBGA) e os derivados sulfatado (LBGS) e carboxilado (LBGC), com carga negativa. O cloreto de glicidiltrimetilamónio foi o agente alquilante para obtenção da LBGA, o complexo de trióxido de enxofre e N,N-dimetilformamida (SO3DMF), o agente sulfatante na síntese da LBGS, e a
2,2,6,6-tetrametilpiperidina-1-oxil foi o agente oxidante na produção da LBGC. Os derivados foram caraterizados por espectroscopia de infravermelho de transformada de Fourier, ressonância magnética nuclear, análise elementar, cromatografia de permeação de gel e difração de raios-X. A intenção de uma aplicação farmacêutica implicou a análise toxicológica dos derivados. A avaliação da atividade metabólica de células Caco-2 após exposição (3 h ou 24 h) à LBG ou aos derivados sintetizados foi realizada por MTT, que mostrou que, com exceção da LBGA, os materiais induziram viabilidades acima dos 70% quando testados em concentrações até 1 mg/mL. Um ensaio complementar que avalia a integridade da membrana celular (libertação de LDH) conferiu resultados semelhantes. Foram produzidas várias formulações de nanopartículas que utilizaram LBGA ou quitosano como polímero carregado positivamente e LBGC ou LBGS como polímero negativo. As nanopartículas foram obtidas com rendimento de produção até 58%, enquanto os tamanhos variaram entre 180 e 830 nm e o potencial zeta entre -28 mV e
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+48 mV, dependendo da composição qualitativa e quantitativa. A caraterização morfológica realizada em algumas formulações (LBGA/LBGS e CSup/LBGS) por microscopia eletrónica de transmissão sugere que as nanopartículas apresentam uma estrutura sólida e compacta e forma aproximadamente esférica. As nanopartículas de CSup/LBGS, posteriormente selecionadas para a associação de antigénio, demonstraram manter a estabilidade físico-química por pelo menos 3 meses quando armazenadas a 4 ºC. As nanopartículas de LBGA/LBGS e CSup/LBGS revelaram ausência de citotoxicidade em células Caco-2 após 3 h e 24 h de exposição, quando em concentrações até 1.0 mg/mL (ensaio MTT), uma observação relevante considerando a forte citotoxicidade do derivado LBGA. O ensaio de libertação de LDH revelou maior citotoxicidade da formulação CSup/LBGS (24 h; 1.0 mg/mL), não observada no ensaio MTT.
Dois antigénios modelo (um extrato celular particulado de Salmonella Enteritidis – HE, e um antigénio solúvel – ovalbumina, OVA) foram associados às nanopartículas CSup/LBGS com eficácia aproximada de 30%. Um estudo de estabilidade revelou ausência de efeito negativo do processo de associação sobre a integridade estrutural do antigénio, mantendo a sua antigenicidade. As nanopartículas exibiram propriedades físico-químicas adequadas para uma aplicação em imunização oral (tamanho de 180 – 200 nm; potencial zeta positivo de 10 – 13 mV) e demonstraram retardar a libertação dos antigénios. Neste sentido, observou-se uma libertação muito limitada de HE em meios gástrico e intestinal simulados, enquanto a OVA libertou no máximo 40% no primeiro meio. Ensaios in vivo incluíram a administração de nanopartículas contendo HE ou OVA a ratinhos BALB/c. Durante cinco/seis semanas após imunização oral e subcutânea, a resposta imunológica sistémica (IgG1 e IgG2a) e mucosa (IgA) foi avaliada. O efeito adjuvante das nanopartículas de CSup/LBGS na resposta imunológica após imunização oral foi demonstrado, apesar de se ter verificado apenas para o antigénio solúvel OVA. Pelo contrário, verificou-se uma ausência de efeito quando se testou HE, um antigénio particulado. As nanopartículas proporcionaram ainda um equilíbrio na resposta Th1/Th2, o que é relevante para uma proteção imunológica eficiente.
De forma geral, o LBGS foi o derivado sintetizado que evidenciou maior capacidade para complexação com quitosano com vista à produção de nanopartículas com propriedades adequadas para imunização oral. Além disso, há uma indicação preliminar do potencial do sistema para imunização oral, apesar de este depender do tipo de antigénio.
Palavras-chave: complexo antigénico de Salmonella Enteritidis, goma de alfarroba,
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Braz, L., Dionísio, M., Grenha, A., 2011. Chitosan-based nanocarriers: effective vehicles for mucosal protein delivery. In: S.P. Davis (Ed.), Chitosan: manufacturing, properties and usage. Nova Science Publishers, New York. p. 365-412
Braz, L., Rodrigues, S., Fonte, P., Grenha, A., Sarmento, B., 2011. Mechanisms of chemical and enzymatic chitosan biodegradability and its application on drug delivery. In: G. Felton (Ed.), Biodegradable Polymers: Processing, Degradation and Applications. Nova Science Publishers, New York. p. 325-364
Braz, L., Grenha, A., Corvo, M., Lourenço, J.P., Ferreira, D., Sarmento, B., Rosa da Costa, A.M. Synthesis and characterization of Locust Bean Gum derivatives and their application in the production of nanoparticles, submitted for publication to Carbohydrate Polymers
Braz, L., Camacho, A., Grenha, A., Ferreira, D., Rosa da Costa, A.M., Gamazo, C., Sarmento, B., Chitosan/Sulfated Locust Bean Gum nanoparticles: In vitro and in vivo evaluation towards an application in oral immunization, submitted for publication to International Journal of Pharmaceutics
Oral communications
Braz, L.; Grenha, A.; Sarmento, B.; Rosa da Costa, A. Novel Locust Bean Gum nanoparticles for protein delivery, XVIII International Conference on Bioencapsulation, Oporto – Portugal, September 2010
Braz, L., Grenha, A., Ferreira, D., Rosa da Costa, A.M., Sarmento, B. Locust Bean Gum based nanoparticulate system for oral antigen delivery. 9th Central European
Symposium on Pharmaceutical Technology, Dubrovnik – Croatia, September 2012
Poster presentations
Braz, L.; Grenha, A.; Sarmento, B.; Rosa da Costa, A. Synthesis of Locust Bean Gum
New Derivatives for Drug Delivery Applications. 70th FIP World Congress of
Pharmacy/Pharmaceutical Sciences, Lisbon – Portugal, August 2010
Braz, L., Grenha, A., Ferreira, D., Rosa da Costa, A.M., Sarmento, B. Modification of a galactomannan-based polysaccharide and application in drug delivery. I Simpósio Nacional de Nanociência e Nanotecnologia Biomédica, Lisbon – Portugal, May 2011
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Braz, L., Grenha, A., Ferreira, D., Rosa da Costa, A.M., Sarmento, B. Cytotoxicity evaluation of Locust Bean Gum derivatives and their application in drug delivery. 3rd
PharmSciFair, Prague – Czech Republic, June 2011
Silva, A., Grenha, A., Rosa da Costa, A.M., Braz, L. Análise multifactorial dos parâmetros que influenciam o rendimento de purificação da goma de alfarroba. IV Congresso Ibero-Americano de Ciências Farmacêuticas, Lisbon – Portugal, June 2011 Braz, L., Grenha, A., Ferreira, D., Rosa da Costa, A.M., Sarmento, B. Locust Bean
Gum derivatives for nanometric drug delivery. 19th Portuguese-Spanish Conference on
Controlled Drug Delivery, Oporto – Portugal, October 2011
Braz, L., Grenha, A., Ferreira, D., Rosa da Costa, A.M., Sarmento, B. Locust Bean Gum based nanoparticles for oral antigen delivery. 2nd International Conference on
Pharmaceutics and Novel Drug Delivery Systems, San Francisco - EUA, February 2012
Braz, L., Camacho, A., Grenha, A., Ferreira, D., Rosa da Costa, A.M., Sarmento, B., Gamazo, C. Salmonella Enteritidis oral immunization mediated by chitosan-sulphated locust bean gum nanoparticles. 11th International Conference of the European Chitin Society, Oporto - Portugal, May 2013
Braz, L., Grenha, A., Ferreira, D., Rosa da Costa, A.M., Sarmento, B.
Ovalbumin-loaded sulphated locust bean gum nanoparticles for oral immunization. 3rd Conference
on Innovation in Drug Delivery: Advances in Local Drug Delivery, Pisa - Italy, September 2013
Braz, L., Grenha, A., Ferreira, D., Rosa da Costa, A.M., Sarmento, B. Locust Bean Gum-based nanoparticles as antigens carriers. Nano2013.pt - II Simpósio Nacional de Nanociência e Nanotecnologia Biomédica, Lisbon – Portugal, October 2013
Braz, L.; Grenha, A.; Ferreira, D.; Rosa da Costa, A.M.; Sarmento, B., Immunization
study of OVA encapsulated in locust bean gum based nanoparticles, 9th World Meeting
on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology, Lisbon - Portugal, April 2014
xvii ABSTRACT ... xi RESUMO ... xiii LIST OF PUBLICATIONS AND COMMUNICATIONS ... xv TABLE OF CONTENTS ... xvii LIST OF FIGURES ... xxi LIST OF TABLES ... xxiv ABBREVIATIONS ... xxv CHAPTER 1 ... 1 1. General introduction ... 3 1.1. Nanoparticles as carriers in drug delivery ... 3 1.1.1. Methods for nanoparticle production ... 5 1.1.2. Materials for nanoparticle production ... 8 1.1.2.1. Locust bean gum ... 11 1.1.2.1. Chitosan ... 13 1.2. Nanoparticle application in oral immunization ... 16 1.2.1. General concepts in immunization ... 16 1.2.2. Oral immunization ... 18 1.2.3. The role of nanoparticles: Locust bean gum as potential adjuvant ... 23 CHAPTER 2 ... 25 2. Motivations and Objectives ... 27 CHAPTER 3 ... 29 3. Synthesis and characterization of Locust Bean Gum derivatives and their application in the production of nanoparticles ... 31 3.1. Introduction ... 31 3.2. Materials and methods ... 33 3.2.1. Materials ... 33 3.2.2. Cell line ... 33
xviii 3.2.3. Synthesis of Locust Bean Gum derivatives ... 34 3.2.3.1. Purification of Locust Bean Gum ... 34 3.2.3.2. Sulfation of Locust Bean Gum ... 34 3.2.3.3. Carboxylation of Locust Bean Gum ... 35 3.2.3.4. Quaternary ammonium salt of Locust Bean Gum ... 36 3.2.4. Chemical characterization of Locust Bean Gum derivatives ... 36 3.2.4.1. Fourier transform infrared (FTIR) spectroscopy ... 36 3.2.4.2. Elemental analysis ... 36 3.2.4.3. Nuclear magnetic resonance (NMR) spectroscopy ... 37 3.2.4.4. GPC/SEC3 analysis ... 37 3.2.4.5. X‐ray diffraction (XRD) ... 37 3.2.5. Production of Locust Bean Gum‐based nanoparticles ... 37 3.2.5.1. CS/LBGS and CS/LBGC nanoparticles ... 38 3.2.5.2. LBGA/LBGS nanoparticles ... 38 3.2.6. Characterization of Locust Bean Gum‐based nanoparticles ... 39 3.2.6.1. Size, polydispersion index and ζ potential ... 39 3.2.6.2. Production yield ... 39 3.2.6.3. Morphological analysis ... 39 3.2.7. Safety evaluation ... 40 3.2.8. Statistical analyses ... 41 3.3. Results and discussion ... 42 3.3.1. Synthesis and chemical characterization of Locust Bean Gum derivatives ... 42 3.3.2. Characterization of nanoparticles ... 52 3.3.2.1. CS/LBGS and CS/LBGC nanoparticles ... 53 3.3.2.2. LBGA/LBGS nanoparticles ... 58 3.3.3. Safety evaluation ... 60 3.4. Conclusions ... 69 CHAPTER 4 ... 71 4. Chitosan/Sulfated Locust Bean Gum nanoparticles: in vitro and in vivo evaluation towards an application in oral immunization ... 73 4.1. Introduction ... 73 4.2. Materials and methods ... 74 4.2.1. Materials ... 74 4.2.2. Cell line... 75 4.2.3. Production of Locust Bean Gum‐based nanoparticles ... 75
xix 4.2.4. Characterization of CSup/LBGS nanoparticles ... 77 4.2.4.1. Size, zeta potential and polidispersion index ... 77 4.2.4.2. Production yield ... 77 4.2.4.3. Association efficiency ... 78 4.2.4.4. Morphological analysis ... 78 4.2.5. In vitro evaluation of CSup/LBGS nanoparticles ... 79 4.2.5.1. Evaluation of the structural integrity and antigenicity of the loaded antigens ... 79 4.2.5.1.1. HE‐loaded CSup/LBGS nanoparticles... 79 4.2.5.1.2. OVA‐loaded CSup/LBGS nanoparticles ... 79 4.2.5.2. Stability evaluation on storage ... 80 4.2.5.3. In vitro release in SGF and SIF ... 80 4.2.5.4. Safety evaluation of unloaded nanoparticles ... 81 4.2.6. In vivo evaluation of the immune response in BALB/c mice ... 82 4.2.6.1. HE‐loaded CSup/LBGS nanoparticles ... 82 4.2.6.2. OVA‐loaded CSup/LBGS nanoparticles ... 83 4.2.7. Statistical analyses ... 84 4.3. Results and discussion ... 84 4.3.1. Characterization of unloaded CSup/LBGS nanoparticles ... 84 4.3.2. Characterization of loaded nanoparticles ... 89 4.3.2.1. HE‐loaded CSup/LBGS ... 89 4.3.2.2. OVA‐loaded CSup/LBGS ... 93 4.3.3. In vitro evaluation of Locust Bean Gum‐based nanoparticles ... 96 4.3.3.1. Evaluation of the structural integrity and antigenicity of the loaded antigens ... 96 4.3.3.2. Stability evaluation on storage ... 98 4.3.3.3. In vitro release in SGF and SIF ... 99 4.3.3.4. Safety evaluation of unloaded nanoparticles ... 101 4.3.4. In vivo evaluation of the immune response in BALB/c mice ... 106 4.3.4.1. HE‐loaded CSup/LBGS nanoparticles ... 106 4.3.4.2. OVA‐loaded CSup/LBGS nanoparticles ... 111 4.4. Conclusions ... 116 CHAPTER 5 ... 119 5. General conclusions ... 121 Future perspectives ... 122
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References ... 125
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of polyelectrolyte complexation. ... 7
Figure 1.2 – Chemical structure of locust bean gum. ... 11 Figure 1.3 – Chemical structure of chitosan. ... 14 Figure 1.4 – Contribution of efficacy, safety, feasibility and cost to the development of
vaccines (118). ... 16
Figure 3.1 – Scheme of the chemical modifications introduced in LBG. ... 44 Figure 3.2 – FTIR spectra of purified Locust Bean Gum (LBG) and its ammonium (LBGA),
carboxylate (LBGC) and sulfate (LBGS) derivatives. ... 45
Figure 3.3 – FTIR spectra of Locust Bean Gum sulfate derivatives (LBGS) obtained in
method 1 (M1) and method 2 (M2). B1, B2 and B3 refer to batch 1, 2 and 3, respectively. ... 47
Figure 3.4 – 1H-NMR of (a) LBG, (b) LBGA, (c) LBGS, and (d) LBGC; the big singlet
centered at 4.7 ppm is due to HOD (identified in grey). ... 49
Figure 3.5 – 13C-NMR of LBGC. ... 50
Figure 3.6 – XRD patterns of (a) pristine locust bean gum (LBG), (b) sulfated LBG
(LBGS), (c) ammonium LBG (LBGA), and (d) carboxylated LBG (LBGC). ... 52
Figure 3.7 – Effect of charge ratio (-/+) on the zeta potential of CS/LBGS nanoparticles. 54 Figure 3.8 – Effect of charge ratio (-/+) on the zeta potential of CS/LBGC nanoparticles. 57 Figure 3.9 – TEM microphotograph of LBGA/LBGS = 1:2 (w/w) nanoparticles. ... 60 Figure 3.10 - Caco-2 cell viability measured by the MTT assay after 3 h exposure to
increasing concentrations of bulk Locust Bean Gum, purified Locust Bean Gum (LBG) and its ammonium (LBGA), carboxylate (LBGC) and sulfate (LBGS) derivatives. Data represent mean ± SEM (n ≥ 3, six replicates per experiment at each concentration). Dashed line indicates 70%. * P < 0.05 compared with DMEM. ... 62
Figure 3.11 - Caco-2 cell viability measured by the MTT assay after 24 h exposure to
increasing concentrations of bulk Locust Bean Gum, purified Locust Bean Gum (LBG) and its ammonium (LBGA), carboxylate (LBGC) and sulfate (LBGS) derivatives. Data represent mean ± SEM (n ≥ 3, six replicates per experiment at each concentration). Dashed line indicates 70%. * P < 0.05 compared with DMEM. ... 62
Figure 3.12 – Caco-2 cell viability measured by the LDH release assay after 24 h
exposure to 1 mg/mL solutions of bulk Locust Bean Gum, purified Locust Bean Gum (LBG) and its ammonium (LBGA), carboxylate (LBGC) and sulfate (LBGS) derivatives. Data represent mean ± SEM (n ≥ 3, three replicates per experiment). * P < 0.05 compared with DMEM. ... 65
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Figure 3.13 – A549 cell viability measured by the MTT assay after 3 h and 24 h exposure
to increasing concentrations of sulfate locust bean gum (LBGS) derivative. Data represent mean ± SEM (n ≥ 3, six replicates per experiment at each concentration). Dashed line indicates 70%. * P < 0.05 compared with respective control (DMEM). ... 66
Figure 3.14 – Calu-3 cell viability measured by the MTT assay after 3 h and 24 h
exposure to increasing concentrations of sulfate locust bean gum (LBGS) derivative. Data represent mean ± SEM (n ≥ 3, six replicates per experiment at each concentration). Dashed line indicates 70%. * P < 0.05 compared with respective control (DMEM). ... 66
Figure 3.15 – Caco-2 cell viability measured by the MTT assay after 3 h exposure to
increasing concentrations of ammonium Locust Bean Gum (LBGA) derivative, sulfate Locust Bean Gum (LBGS) derivative and LBGA/LBGS nanoparticles (NP). Data represent mean ± SEM (n ≥ 3, six replicates per experiment at each concentration). Dashed line indicates 70%. * P < 0.05 compared with DMEM. ... 67
Figure 3.16 – Caco-2 cell viability measured by the MTT assay after 24 h exposure to
increasing concentrations of ammonium Locust Bean Gum (LBGA) derivative, sulfate Locust Bean Gum (LBGS) derivative and LBGA/LBGS nanoparticles (NP). Data represent mean ± SEM (n ≥ 3, six replicates per experiment at each concentration). Dashed line indicates 70%. * P < 0.05 compared with DMEM. ... 68
Figure 4.1 – Effect of charge ratio (-/+) on the zeta potential of CSup/LBGS nanoparticles.
... 87
Figure 4.2 - TEM microphotograph of CSup/LBGS 1:2 (w/w) nanoparticles. ... 88 Figure 4.3 – TEM microphotograph of HE-loaded CSup/LBGS (1:2, w/w; 8% HE)
nanoparticles. ... 93
Figure 4.4 – Immunoblot (a) and SDS-PAGE (b) analyses of free HE (1) and HE released
from HE-loaded CSup/LBGS nanoparticles (2) (PS: standard proteins). ... 96
Figure 4.5 – SDS-PAGE (a) and immunoblot (b) analyses of free OVA (1) and OVA
released from OVA-loaded CSup/LBGS nanoparticles (2) (PS: standard proteins). ... 97
Figure 4.6 – Size (square marks) and zeta potential (triangular marks) evolution as
function of time upon storage at 4 ºC of CSup/LBGS 1:2 (w/w) unloaded nanoparticles (empty marks) and HE-loaded nanoparticles (8% w/w; filled marks); (mean ± SD, n ≥ 3). ... 99
Figure 4.7 – Antigen released overtime from CSup/LBGS nanoparticles in simulated
gastric fluid (SGF) and simulated intestinal fluid (SIF), at 37 ºC (mean ± SD; n ≥ 3). ... 100
Figure 4.8 – Caco-2 cell viability measured by the MTT assay after 3 h exposure to
increasing concentrations of Chitosan (CSup), sulfate Locust Bean Gum (LBGS) derivative and CSup/LBGS nanoparticles (NP). Data represent mean ± SEM (n ≥ 3, six
xxiii
increasing concentrations of Chitosan (CSup), sulfate Locust Bean Gum (LBGS) derivative and CSup/LBGS nanoparticles (NP). Data represent mean ± SEM (n ≥ 3, six replicates per experiment at each concentration). Dashed line indicates 70%. * P < 0.05 compared with DMEM. ... 104
Figure 4.10 – Caco-2 cell viability measured by the LDH release assay after 24 h
exposure to 1 mg/mL solutions of Chitosan (CSup), sulfate Locust Bean Gum (LBGS) derivative and CSup/LBGS nanoparticles (NP). Data represent mean ± SEM (n ≥ 3, three replicates per experiment). * P < 0.05 compared with DMEM. ... 105
Figure 4.11 – Immunogenicity of HE after oral administration in mice. Serum A) IgG1 and
B) IgG2a systemic response, and C) IgA mucosal response after oral immunization of 5 female BALB/c mice with 200 µg of HE solution (HE), 200 µg of encapsulated HE (NP-HE) and the corresponding mass of blank nanoparticles (NP). In the HE group the results of one mouse were rejected due to the high initial absorbance (week 0) (mean ± SEM; n ≥ 4). ... 108
Figure 4.12 – Immunogenicity of HE after S.C. administration in mice. Serum A) IgG1 and
B) IgG2a systemic response, and C) IgA mucosal response after S.C. immunization of 5 female BALB/c mice with 40 µg of HE solution (HE), 40 µg of encapsulated HE (NP-HE) and the corresponding mass of blank nanoparticles (NP) (mean ± SEM; n = 5). ... 110
Figure 4.13 – Immunogenicity of OVA after oral administration in mice. Serum anti-OVA
A) IgG1; B) IgG2a and C) faecal anti-OVA IgA response in BALB/c mice (n = 6) after oral immunization with 100 µg of OVA solution (OVA) or 100 µg of encapsulated OVA (NP-OVA). Antibody titers were determined in pooled serum samples at days 0, 7, 14, 21 and 28 post-administration. ... 113
Figure 4.14 – Immunogenicity of OVA after S.C. administration in mice. Serum anti-OVA
A) IgG1; B) IgG2a and C) faecal anti-OVA IgA response in BALB/c mice (n = 6) after S.C. immunization with 20 µg of OVA solution (OVA) or 20 µg of encapsulated OVA (NP-OVA). Antibody titers were determined in pooled faecal samples at days 0, 7, 14, 21 and 28 post-administration. ... 115
xxiv
LIST OF TABLES
Table 1.1 - Advantages and limitations of nanoparticles for drug delivery applications. .... 5 Table 1.2 – Pros and cons of polysaccharides as nanoparticle matrix materials (53). ... 9 Table 1.3 – Advantages and disadvantages of oral administration (141). ... 19 Table 1.4 – Toll-like receptors (TLRs) and their specific ligands (150)... 20 Table 1.5 – Some of the most frequent C-type lectins and their specific ligands (152, 153).
... 21
Table 3.1 – Elemental analysis data from the sulfate (LBGS), carboxylate (LBGC) and
ammonium (LBGA) derivatives of locust bean gum (LBG). ... 46
Table 3.2 – GPC analysis of purified Locust Bean Gum (LBG), and its ammonium
(LBGA), carboxylate (LBGC) and sulfate (LBGS) derivatives. ... 51
Table 3.3 - Physicochemical characteristics and production yield of CS/LBGS unloaded
nanoparticles (mean ± SD; n ≥ 3). Different letters represent significant differences in each parameter (P < 0.05). ... 53
Table 3.4 - Physicochemical characteristics and production yield of CS/LBGC unloaded
nanoparticles (mean ± SD; n ≥ 3). Different letters represent significant differences in each parameter (P < 0.05). ... 56
Table 3.5 - Physicochemical characteristics and production yield of LBGA/LBGS unloaded
nanoparticles (mean ± SD; n ≥ 3). Different letters represent significant differences in each parameter (P < 0.05). ... 58
Table 4.1 - Physicochemical characteristics and production yield of CSup/LBGS unloaded
nanoparticles (mean ± SD; n ≥ 3). Different letters represent significant differences in each parameter (P < 0.05). ... 85
Table 4.2 – Physicochemical characteristics, production yield and association efficiency of
HE antigens in CSup/LBGS nanoparticles (mean ± SD; n ≥ 3). Different letters represent significant differences in each parameter, evaluated separately for each mass ratio (P < 0.05). ... 91
Table 4.3 – Physicochemical characteristics, production yield end association efficiency of
OVA in CSup/LBGS nanoparticles (mean ± SD; n ≥ 3). Different letters represent significant differences in each parameter (P < 0.05). ... 95
xxv AE: Association efficiency
AIDS:Acquired immune deficiency syndrome
ANOVA: One-way analysis of variance APCs: Antigen-presenting cells
AS03: Adjuvant system 03 AS04: Adjuvant system 04 AUC: Area under the curve BCA: Bicinchoninic acid
BCG: Bacillus Calmette-Guérin BSA: Bovine serum albumin CD: Cluster of differentiation CD4+: CD4 positive T cell
CD8+: CD8 positive T cell
CpG DNA: Cytosine – phosphate – guanine deoxyribonucleic acid CS: Chitosan
CSup: Ultrapure chitosan DCs: Dendritic cells
DC-SIGN: Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin DMEM: Dulbecco’s modified Eagle’s medium
DMF: Dimethylformamide DMSO: Dimethyl sulfoxide DNA: Deoxyribonucleic acid
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DO: Degree of oxidation DS: Degree of substitution
dsRNA: Double-stranded ribonucleic acid EDTA: Ethylenediamine tetraacetic acid ELISA: Enzyme-linked immunosorbent assay FAE: Follicle associated epithelium
FBS: Fetal bovine serum
FDA: Food and drug administration FTIR: Fourier transform infrared GALT: Gut-associated lymphoid tissue GlcNAc: N-acetylglucosamine
GPC/SEC3: Triple detection gel permeation chromatography
GRAS: Generally recognized as safe
GTMAC: N-glycidyl-N,N,N-trimethylammonium chloride
HE: Immunogenic subcellular extract obtained from whole Salmonella Enteritidis HPLC: High performance liquid chromatography
ICAM-3: Intercellular adhesion molecule 3 IFN-γ: Interferon-gamma
IgA: Immunoglobulin A IgG1: Immunoglobulin G1 IgG2a: Immunoglobulin G2a IL-10: Interleukin 10
IL-17: Interleukin 17 IL-2: Interleukin 2
xxvii IL-6: Interleukin 6
IPN: Interpenetrating polymer network
ISO: International Organization for Standardization LBG: Locust bean gum
LBGA: Locust bean gum ammonium derivative LBGC: Locust bean gum carboxylate derivative LBGS: Locust bean gum sulfate derivative LC: Loading capacity
LDH: Lactate dehydrogenase LPS: Lipopolysaccharide M cells: Microfold cells
M/G: Mannose/galactose ratio ManNAc : N-acetylmannosamine MBL: Mannose-binding lectin
MF59: Oil in water emulsion composed of squalene, polysorbate 80, sorbitan trioleate and citrate buffer
MHC I: Major histocompatibility complex I MHC II: Major histocompatibility complex II
Mn: Number average molecular weight
MPLA: Monophosphoryl lipid A
MTT: Thiazolyl blue tetrazolium bromide
xxviii
NMR: Nuclear magnetic resonance NP: Nanoparticles
OD: Optical density OVA: Ovalbumin
PAMP: Pathogen-associated molecular pattern PBS: Phosphate buffered saline
PBS-T: Phosphate buffered saline-tween PdI: Polydispersion index
PLGA: Poly(d,l-lactide-co-glycolide) PPs: Peyer's patches
PRR: Pattern recognition receptors PS: Proteins standard
PVA: Polyvinyl alcohol PY: Production yield
Rg: Radius of gyration
SDS: Sodium dodecyl sulfate
SDS-PAGE: Sodium dodecyl sulfate - polyacrylamide gel electrophoresis SGF: Simulated gastric fluid
SIF: Simulated intestinal fluid
SO3.DMF: N,N-Dimethylformamide sulfur trioxide
SP-A: Surfactant protein A SP-D: Surfactant protein D
ssRNA: Single-stranded ribonucleic acid TCRs: T cell receptors
xxix TGF-β: Transforming growth factor β
Th: T helper Th1: T-helper type 1 Th17: T-helper type 17 Th2: T-helper type 2 Th3: T-helper type 3 TLR: Toll-like receptor XRD: X-ray diffraction
1
CHAPTER 1
GENERAL INTRODUCTION
The information presented in this chapter was partially published in the following publications:
Braz, L., Dionísio, M., Grenha, A., 2011. Chitosan-based nanocarriers: effective vehicles
for mucosal protein delivery. In: S.P. Davis (Ed.), Chitosan: manufacturing, properties and usage. Nova Science Publishers, New York. p. 365-412.
Braz, L., Rodrigues, S., Fonte, P., Grenha, A., Sarmento, B., 2011. Mechanisms of
chemical and enzymatic chitosan biodegradability and its application on drug delivery. In: G. Felton (Ed.), Biodegradable Polymers: Processing, Degradation and Applications. Nova Science Publishers, New York. p. 325-364.
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3
1. General introduction
1.1. Nanoparticles as carriers in drug delivery
The recent decades have brought to the market many new biomolecules that have been identified as having therapeutic potential. This boom is directly related with advances in the biotechnological industry, making available a very considerable number of molecules that are protein-based. These molecules are usually called biopharmaceuticals, meaning that they are biological in nature and manufactured using biotechnology (1). A considerably wide variety of molecules is included in this group, from protein and peptides, to antigens and nucleic acids. In many cases their promise is so high that they are thought to occupy in the future an undisputed place alongside other established therapies. Although therapeutically promising, biopharmaceuticals are very instable compounds and their administration is extremely challenging, due to inherent physicochemical and biopharmaceutical properties (2, 3). This is the main reason why parenteral delivery frequently represents the unique administration possibility, as is often verified for vaccines, for example. Nevertheless, the parenteral route involves an invasive and painful administration, thus not being easily accepted by the patients and many times leading to therapeutic incompliance (3, 4). A gap is, thus, clearly identified which needs to be filled, compelling researchers to invest in this area in order to find adequate alternatives that permit effective, safe, cheap and comfortable administration of biopharmaceutical molecules through other routes. Comparing with the modality of parenteral delivery, mucosal administration is advantageous as systemic pathway, mainly because it is non-invasive, reducing patient discomfort, but also because it generally does not require the involvement of skilled personnel for the administration, thus reducing the costs of the process (3, 5). In this manner, the development of non-injectable delivery systems for mucosal administration could enhance significantly patient’s compliance, thereby leading to increased therapeutic benefits.
The therapeutic action of proteins and protein-based molecules is not only limited by the potential degradation in biological environments, but also compromised by their low ability to reach the therapeutic site of action (3, 6, 7). In fact, these limitations are related either with the presence of a great number of functional groups susceptible of chemical degradation, or with the high hydrophilic character of the proteins, which results in poor permeability (8, 9). In addition, drug delivery via mucosal routes faces other major restrictions, including specific mechanisms of defense, the possibility to induce immune reactions at the delivery site and, generally, limitations in the surface area available for
4
absorption (10). As such, a meaningful challenge for current pharmaceutical scientists has been the need to develop suitable vehicles that permit delivering macromolecules through alternative routes of administration. These drug delivery carriers should exhibit a sort of characteristics, including capacity for high drug association and the ability to enhance drug physicochemical stability, providing protection to encapsulated drugs from the moment of carrier production until release. Furthermore, in many cases, the carriers are expected to regulate the drug release profile, while allowing an intimate contact of molecules with mucosal barriers, contributing for their epithelial permeation. In such a task, there is a consensus in that the materials and methods used to prepare the referred vehicles play relevant roles (11, 12).
Directing the research efforts towards the development of adequate vehicles for the purpose of drug delivery through distinct routes of administration, resulted in the appearance of several drug delivery systems like nanoparticles and microspheres. Nanoparticulate-based technologies have reached a position of evidence and nanoparticles are one of the most approached systems. The International Organization for Standardization (ISO) defines nanoparticles as particles with at least one dimension less than 100 nm (13). This definition is however not consensual in the field of drug delivery
and there are many authors considering that nanoparticles are carriers with dimensions between 10 and 1000 nm (14-17). The interest in nanosystems (submicron sized systems) relies on several differentiating features that include an increased surface-to-volume ratio, in many cases displaying surface functionality, which offers high potential for the association of biopharmaceuticals (15). Biological transport processes have been reported to be affected by the physical attributes of nanocarriers, both anatomically and down to the cellular ad subcellular levels (18). Actually, nanocarriers have been reported to increase drug absorption by reducing the resistance of the epithelium to drug transport in a localized area or by carrying the drug across the epithelium (19). In this regard, transport has been described as more favorable for nanoparticles than for microparticles (2, 20, 21). In addition, colloidal carriers are reported to have improved capacity to interact with mucosal epithelial membranes (20), maximum interaction being reported to occur for systems within 50 – 500 nm (8). Colloidal carriers have also shown several times the ability to control the drug release profile of encapsulated molecules (22-24). Importantly, specifically regarding the delivery of biopharmaceuticals, an improvement of molecule stability, bioavailability, targeting, uptake and biological activity has been shown (24-26).
Table 1.1 summarizes the advantages and drawbacks of nanoparticles for drug delivery
5
Table 1.1 - Advantages and limitations of nanoparticles for drug delivery applications. Advantages Limitations
High surface/volume ratio Undefined physical shape
Ease of surface modification Limited capacity to co-associate other
functional molecules
Maximised contact with mucosae Unknown toxicity profile
High drug concentration in desired site Lack of suitable large-scale production methods
Ability to enter cells Low stability in some biological fluids
Protection of encapsulated molecules Tendency for aggregation
Possibility to provide controlled release Limited loading capacity (unsuitable for less potent drugs)
Possibility of targeted delivery Small size can provide access to
unintended environments
1.1.1. Methods for nanoparticle production
Although rather different materials may be used to produce nanoparticles, including metals and ceramics, polymers have been one of the most used of the material classes. The literature describes many methods to produce polymeric nanoparticles. Naturally, each method has its own pros and cons, and the choice of a particular methodology will mainly depend on specific characteristics of the drug to be encapsulated and the material to be used as matrix (3). The techniques may be categorized into top-down or bottom-up approaches. Top-down processes involve the size-reduction of large particles to the nanometre range, which can be achieved by milling or high pressure homogenization. These processes have much lower application when compared with bottom-up techniques, which involve the assembly of molecules in solution to form defined nanostructures (27, 28). However, it is important to mention that delivery systems resulting from bottom-up technologies usually display size polydispersity (29), which in some cases limits nanoparticle usefulness. In fact, it is assumed that in a polydisperse system, larger nanoparticles might have higher drug loading capacity, while smaller nanoparticles are expected to have higher efficiency at delivering drugs to tissues or cells.
6
In a limit situation, this means that, even if the drug carrier has high association efficiency, the efficacy of the delivery may be poor (30). Therefore, fabrication processes should be rigorously optimized to provide a compromise between satisfactory association efficiency and the most suitable size for the established objective. Interestingly, a recent technological development related to a top-down process termed particle replication in non-wetting templates, which is a modified soft lithography technique, has demonstrated independent control over nanoparticle size, as well as other parameters that include shape, modulus (stiffness) and surface chemistry (29, 31).
Bottom-up techniques might also be classified according to whether the formulation of nanoparticles involves a polymerization reaction or is achieved directly from a preformed polymer (32). Methods involving polymerization are divided in emulsion polymerization and interfacial polymerization (32). When nanoparticles are prepared from preformed polymers, which is the most common approach, the diversity of techniques increases, involving methods based on emulsification, polymer desolvation, or intermolecular electrostatic interactions (11, 32). Contrary to low molecular weight drugs, biopharmaceuticals possess organized structures (proteins for example may have secondary, tertiary, or even quaternary structure) with labile bonds and side chains of chemically reactive groups. This specific structure defines the exact properties and activities of the molecules and, therefore, it is crucial to ensure its preservation during the association procedures, as its disruption or the modification of side chains can lead to loss of activity of the molecule. This fragile nature requires that methods selected for association do not damage the molecule structure, reduce their biological activity, or render them immunogenic (3).
Methods based on the establishment of intermolecular electrostatic interactions are one of the most reported and are applied when the matrix of nanoparticles is composed by at least one polyelectrolyte, that is, a polymer that exhibits charged groups when in solution. The principle of this methodology is the ability of polyelectrolytes to establish stable links with oppositely charged groups (33). Two different methods are described based on electrostatic interaction. Ionic gelation is the used denomination when the polyelectrolyte is a polymer with gelling ability (such as chitosan or alginate, for instance) and its gelation is induced by small anionic molecules, such as phosphate, citrate and sulfate groups. A very typical example is that of chitosan nanoparticles prepared by interaction of chitosan amino groups with the phosphate groups of tripolyphosphate (34). In turn, polyelectrolyte
complexation is the name of the technique when the groups mediating the interaction are
provided by two oppositely charged polyelectrolytes, instead of involving one small molecule (35). The latter approach is often referred to as complex coacervation or
7
interfacial coacervation (36, 37), and includes for example chitosan/alginate (38), chitosan/carrageenan (39) or chitosan/dextran sulfate (40) nanoparticles. Figure 1.1 displays a schematic representation of the method of polyelectrolyte complexation to produce nanoparticles. The assembly of nanoparticles is easily and immediately observed upon pouring a solution of one polyelectrolyte over a solution of the oppositely charged polyelectrolyte, under mild stirring.
The popularity of the method is mainly due to the fact that it usually involves a complete hydrophilic environment and mild preparation conditions (41), avoiding the use of organic solvents or high shear forces and making association of labile drugs, such as biopharmaceuticals, an easier task (33, 42).
Figure 1.1 – Schematic representation of the preparation of nanoparticles by the method
of polyelectrolyte complexation.
The literature indicates that, in order to obtain nanocarriers with pre-established characteristics by this method, an optimization of the process should be performed
8
focusing aspects such as the concentration of polyelectrolytes, the stirring conditions and the conditions of centrifugation (43).
1.1.2. Materials for nanoparticle production
The selection of appropriate materials for drug delivery approaches should be driven by several requirements, including biocompatibility, biodegradability, versatility and low overall costs of production (11, 44). As mentioned above, polymeric nanoparticles are one of the most representative of nanomedicines, as polymers are among the most versatile building units, permitting an easy tailoring of their properties to meet specific requirements (44). By definition, polymeric nanoparticles can be produced using either synthetic or natural polymers. The former are reported frequently, but in many cases they display unsatisfactory biocompatibility, which limits potential clinical applications (45). On the contrary, natural polymers comply more easily with the requirements mentioned above (46). Actually, it has been claimed that one of the ways of avoiding the potential toxicity of nanocarriers, an issue believed to be one of the most relevant in preventing diverse clinical applications so far, may be using natural materials (45). Moreover, these have some remarkable merits over synthetic ones, namely improved capacity for cell adhesion and mechanical properties similar to natural tissues (47).
The class of natural polymers is divided in proteins and polysaccharides. The latter have found a wide range of applications, as there is an extensive variety of materials available for exploration, comparing with proteins. Furthermore, many polysaccharides are obtained from plants or marine organisms, therefore being less probable to induce adverse immunological reactions, as compared with proteins (44). Polysaccharides are complex carbohydrates, composed of monosaccharides joined together by glycosidic bonds (44, 48). The most common basic units composing these carbohydrate polymers include several monosaccharides such as glucose, fructose, galactose, L-galactose, D-mannose, L-arabinose and D-xylose. Some polysaccharides comprise in their structure simple sugar acids (glucuronic, mannuronic and iduronic acids) and also monosaccharide derivatives, like the amino sugars D-glucosamine and D-galactosamine, as well as their derivatives N-acetylneuraminic acid and N-acetylmuramic acid (49). The presence of several of these sugar units on the side chain of carbohydrate polymers makes them good candidates for targeted delivery by carbohydrate recognizing receptors found on the surface of several cells (50, 51).
9
Polysaccharides might have algal, plant, microbial or animal origin, but they all share general properties of natural polymers, including the propensity for biocompatibility, low cost and hydrophilicity (45, 48, 52). Table 1.2 presents the pros and cons of polysaccharides as nanoparticle matrix materials.
Table 1.2 – Pros and cons of polysaccharides as nanoparticle matrix materials (53). Advantages Limitations
Structural flexibility, stability Inter-batch variability
Low cost Limited availability (if widely used)
Bioadhesion capacity Complex and varied composition
Hydrophilicity Difficult to process
Biocompatibility, biodegradability Immunogenicity
Importantly, polysaccharides are economical, readily available, usually biodegradable and with few exceptions, also biocompatible (46, 54). These are the reasons justifying that they assume a relevant role as matrix materials for drug delivery systems and, namely, nanoparticles. Owing to the potential to exhibit biodegradability, carriers based on polysaccharides are expected to be easily eliminated from the organism, as a consequence of their metabolization into small sugar units that integrate conventional metabolic processes, thereby permitting elimination or re-absorption (45). Polysaccharides present diverse physicochemical properties, deriving from multiple chemical structures that also translate into different molecular weights (Mw) and intrinsic
characteristics. Ionic nature, for instance, is one of the greatest items of variation, as cationic, anionic and neutral polysaccharides can be found (46, 48). The hydrophilic character is particularly important, as it allows the production of nanoparticles by methods not involving organic solvents. Additionally, polysaccharides also benefit from a great structural flexibility, forming either linear or branched structures and easily permitting chemical modifications (44, 55). The more sophisticated applications of these polymers to produce nanoparticles, which include controlled or triggered release, or even targeted delivery, frequently demand chemical modifications of polysaccharides. These usually encompass the introduction of ionic or hydrophobic groups, degradable bonds, spacers or targeting moieties (44). The ability to adhere to biological surfaces comprises a relevant
10
advantage in drug delivery applications (48), as these frequently imply an interaction with cell surfaces. In this regard, the reactive functional groups of polysaccharides allow the formation of non-covalent bonds with cell surfaces, affording enhanced residence time and, consequently, increased drug absorption (45, 52). Notwithstanding the relevance of the advantages mentioned above, some drawbacks should also be taken into account, such as the possibility of generating immunogenic responses and the polymer variability related to origin and supplier (47). Regarding the latter, plant-derived polysaccharides pose potential challenges, as structural differences might occur according to the location and plant collection season (46).
As understood from what is described above, although polysaccharides have been traditionally included in formulations as inert materials, modern pharmaceutical design involves these excipients in increasingly relevant roles. In this manner, their application usually intends to endow the dosage forms with multi-functional abilities, such as controlled release, stabilization, emulsification or bioadhesiveness, among others (46, 48). Nevertheless, the set of polysaccharide properties confers a relevant versatility that adds to a wide range of biological abilities, thus generally contributing to an increased application in drug carrier production.
The application of polysaccharides in the production of nanoparticles by polyelectrolyte complexation or ionic gelation is widely reported (34, 38-40, 56, 57). As these methods require hydrophilic materials that exhibit opposite charges, in addition to an absence of toxicity, chitosan is the only natural polycationic polysaccharide that satisfies these needs (52). On the contrary, there are many negatively charged polysaccharides that can be used for this end, including alginate, hyaluronic acid, dextran sulfate, chondroitin sulfate and carrageenan, among many others. However, the interest on using other materials that do not exhibit charge has also been demonstrated occasionally and, in this regard, the synthesis of charged derivatives of these polysaccharides has been reported to serve the strategy, as described for pullulan (58, 59).
Below, the general characteristics of the polysaccharides locust bean gum and chitosan, which were used in this work to produce nanoparticles by polyelectrolyte complexation, are detailed.
11
1.1.2.1. Locust bean gum
Locust bean gum (LBG) is a non-starch polysaccharide, mainly comprised of a high molecular weight (approximately 50 000 – 3 000 000 Da) neutral galactomannan consisting in a linear chain of (1-4)-linked -D-mannopyranosyl units with (1-6)-linked -D-galactopyranosyl residues as side chains (60), as depicted in Figure 1.2.
Figure 1.2 – Chemical structure of locust bean gum.
The polysaccharide is extracted from the seeds of the carob tree (Ceratonia siliqua), where it acts as a reserve material utilized during germination. It is also referred in the literature by several other synonyms, such as carob bean gum, carob seed gum, carob flour or even ceratonia (61) and consists in a white to yellowish white, nearly odorless, powder. The carob is a large tree that grows to about 10 m high in 10-15 years, and starts to bear good quantities of pods around this age, although it may not be fully grown until it is 50 years old. It is very abundant in the Mediterranean region, although its location also extends to various regions of North Africa, South America, and Asia. Its fruit is a long brown pod containing very hard brown seeds, the kernels, which represent approximately 10% of the weight of the fruit. In the processing of carob gum, these seeds are first dehusked by treating the kernels with dilute sulfuric acid or with a thermal mechanical treatment. The seeds are then split lengthwise and the germ portion is separated from the endosperm. Following, the isolated endosperm (42-46% of the seed weight) undergoes grinding, sifting, grading, and packaging (native LBG). The gum may still be simply washed with ethanol or isopropanol to control the biological load (washed LBG) or be further clarified by dispersion in hot water, recovery by precipitation with isopropanol or ethanol, filtering, drying and milling (clarified LBG). The clarified gum has higher galactomannan content, and no longer contains the cell structure. The commercial samples of LBG contain approximately 80-85% galactomannan, 5-12% moisture, 1.7-5%
12
acid-soluble matter, 0.4-1% ash, and 3-7% protein; the samples of clarified LBG contain approximately 3-10% moisture, 0.1-3% acid-soluble matter, 0.1-1% ash, and 0.1-0.7% protein (60, 62-64).
Galactomannans include several polysaccharides, such as LBG, guar gum and tara gum, which mainly differ in the mannose/galactose (M/G) ratio and the substitution pattern of side-chain units. The M/G ratio varies depending on the distribution of the galactose units over the mannose backbone, being approximately 4:1 for LBG, as results from reported mannose and galactose contents varying within 73-86% and 27-14%, respectively (60, 65). This is an approximate ratio, as it is strongly affected by the varying origins of the polymer and plant growth conditions during production (46). This ratio is the main characteristic affecting galactomannans solubility, as higher water solubility is afforded by higher galactose content (66), because it introduces an entropic and a steric barrier to the ordered packing of mannose segments that leads to aggregate formation (67). Galactose grafts to the mannose chain are known to not be spaced regularly, instead assuming random locations on the linear backbone and leading to low-substituted (“smooth”) and densely-substituted (“hairy”) zones (68). These low-substituted blocks of the backbone permit, consequently, the formation of strong intramolecular hydrogen bonds that reduce the hydration of the gum (69). Displaying an M/G ratio of approximately 4:1, LBG presents limited solubility, having propensity to form aggregates in cold water, as the long segments of unsubstituted mannose, which can be as large as 50 mannose units, are prone to undergo aggregation (47, 67, 69). LBG forms very viscous solutions at relatively low concentrations (70). When in solution, galactomannans have an extended rod-like conformation and occupy a large volume of gyration. In a process dependent on the molecular weight, these gyrating molecules collide with each other and with clusters of solvent molecules to produce solutions of high viscosity (69). Being non-ionic in nature, LBG viscosity and solubility are little affected by pH changes within the range of 3-11, as well as by the addition of salts (62, 63). In this context, chemical modifications of galactomannans to perform carboxylation, hydroxylalkylation and phosphorylation have been approached to overcome solubility limitations (71, 72).
Plant resources are renewable and, therefore, one of their important advantages relies on the possibility to have constant material supply, which is ensured if the plants are cultivated or harvested in a sustainable manner. However, plant-based materials also pose potential challenges that include the production of small quantities that may present structural differences depending on the location of the plants from which they originate, as well as the collecting season. In this context, several studies have evidenced that the chemical structure and molecular weight of LBG vary systematically with the type of
